Compositions and methods that modulate cholesterol in a companion animal

ABSTRACT

Cholesterol, high-density lipoproteins, and low-density lipoproteins in a companion animal can be improved by adjusting the diet of the animal to increase the amount of a compound which positively or negatively modulates the cholesterol, high-density lipoproteins, or low-density lipoproteins or adjusting the diet of the animal to decrease the amount of a compound which positively or negatively modulates cholesterol, high-density lipoproteins, or low-density lipoproteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/434,553 filed Dec. 15, 2016, the disclosure of which isincorporated in its entirety herein by this reference.

BACKGROUND

Animals, particularly companion animals (e.g., dogs and cats), maydevelop hypercholesterolemia, an abnormal condition characterized byelevated serum cholesterol levels. Hypercholesterolemia may occurtogether with chronic renal failure, hypothyroidism, cholestasis,hyperadrenocorticoidism, diabetes mellitus, idiopathichypercholesterolemia, lipoprotein lipase deficiency, and primaryhyperchylomicronemia, for example.

Hypercholesterolemia is one of the major causes of atherosclerosis orcoronary heart diseases. Hyperlipidemia is characterized by abnormallyelevated levels of any or all lipids and/or lipoproteins in the blood.Disorders associated with hyperlipidemia and/or hypercholesterolemiainclude cardiovascular disorders. Cardiovascular disorders are a groupof disorders of the heart and blood vessels and include coronary heartdisease, cerebrovascular disease, peripheral arterial disease, rheumaticheart disease, deep vein thrombosis and pulmonary embolism, etc.

Accordingly, it is beneficial to be able to lower the serum cholesterollevels.

SUMMARY

The present disclosure relates generally to pet food compositions;methods of enhancing nutritional benefit of a pet food; and methods formodulating cholesterol, high-density lipoproteins, and/or low-densitylipoproteins in a companion animal. Specifically, the present disclosurerelates to metabolites for modulating at least one of cholesterol,high-density lipoproteins, or low-density lipoproteins in a companionanimal.

The present inventors have developed a predictive model of cholesterol,high-density lipoproteins, and low-density lipoproteins by identifyingmetabolite compounds which correlate to cholesterol, high-densitylipoproteins, or low-density lipoproteins. A very controlled study wasemployed to minimize other external factors by using multiple caninesall fed the same diet. A validation model was then developed by feedingdifferent levels of the identified compounds (via a dietary change) to agroup of canines and measuring changes in the correspondingcholesterol/lipid parameter.

Accordingly, in a general embodiment, the present disclosure provides apet food composition comprising: protein, carbohydrates, fat, fiber, anda metabolite for modulating at least one of cholesterol, high-densitylipoproteins, or low-density lipoproteins in a companion animal. In oneembodiment, the pet food composition can provide at least a 5% increaseor a 5% decrease in the amount of cholesterol, high-densitylipoproteins, or low-density lipoproteins present in the companionanimal. In one aspect, the pet food can provide an increase. In anotheraspect, the pet food can comprise a decrease.

The present disclosure also provides methods of minimizing costsassociated with production of a pet food; methods of enhancingnutritional benefit of a pet food; and methods for modulating the amountof cholesterol, high-density lipoproteins, or low-density lipoproteinsin a companion animal.

An advantage of one or more embodiments provided by the presentdisclosure is to increase or decrease cholesterol, high-densitylipoproteins, or low-density lipoproteins in a companion animal byadjusting the diet of the animal to increase the amount of a compoundwhich positively modulates the specific cholesterol/lipoprotein or todecrease the amount of a compound which negatively modulates thespecific cholesterol/lipoprotein.

Additional features and advantages are described herein and will beapparent from the following detailed description.

DETAILED DESCRIPTION Definitions

As used in this disclosure and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition” or“the composition” includes two or more compositions. The term “and/or”used in the context of “X and/or Y” should be interpreted as “X,” or“Y,” or “X and Y.” Where used herein, the terms “example” and “such as,”particularly when followed by a listing of terms, are merely exemplaryand illustrative, and are not exclusive or comprehensive.

As used herein, “about” is understood to refer to numbers in a range ofnumerals, for example the range of −10% to +10% of the referencednumber, within −5% to +5% of the referenced number, or in one aspect,within −1% to +1% of the referenced number, and in a specific aspect,within −0.1% to +0.1% of the referenced number. Furthermore, allnumerical ranges herein should be understood to include all integers,whole or fractions, within the range. Moreover, these numerical rangesshould be construed as providing support for a claim directed to anynumber or subset of numbers in that range. For example, a disclosure offrom 1 to 10 should be construed as supporting a range of from 1 to 8,from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and soforth.

All percentages expressed herein are by weight of the total weight ofthe food composition unless expressed otherwise. When reference is madeto the pH, values correspond to pH measured at 25° C. with standardequipment. An “amount” can be the total amount of the referencedcomponent per serving of the composition or per distinct unit of thecomposition and/or can be the weight percentage of the referencedcomponent by dry weight. Moreover, an “amount” includes zero; forexample, the recitation of an amount of a compound does not necessarilymean that the compound is present, unless followed by a range thatexcludes zero.

The terms “food,” “food product” and “food composition” mean a productor composition that is intended for ingestion by an animal, including ahuman, and provides at least one nutrient to the animal. Further in thisregard, these terms mean that the product or composition is in a formready for consumption and is not merely an intermediate from which aconsumable product or composition is made, although other foodcompositions can be added in some embodiments. The term “pet food” meansany food composition intended to be consumed by a pet. The term “pet”means any animal which could benefit from or enjoy the compositionsprovided by the present disclosure. For example, the pet can be anavian, bovine, canine, equine, feline, hircine, lupine, murine, ovine,or porcine animal, but the pet can be any suitable animal.

The term “companion animal” means a dog or a cat. In an embodiment, thecompositions and methods disclosed herein involve a senior dog or asenior cat. Dogs are considered senior in the last 25% of their lives.The life span of a dog depends on its size and/or its breed, but for thepresent disclosure a senior dog is a dog that is at least 5 years of age(e.g., at least 6 years of age, at least 7 years of age, or at least 8years of age). The life span of a cat also depends on its size and/orits breed, but for the present disclosure a senior cat is a cat that isat least 7 years of age (e.g., at least 8 years of age, at least 9 yearsof age, or at least 10 years of age).

As used herein, “metabolite” refers to a compound having biologicalactivity in a companion animal that is an intermediate or product ofmetabolism, and includes precursors thereof. As used herein, “precursor”refers to any compound that metabolizes to a metabolite duringmetabolism in a companion animal. For example, if the specificmetabolite is cysteine, “the metabolite” comprises a cysteine precursor(e.g., methionine).

As used herein, “high-density lipoproteins” refers to a type oflipoprotein that transports fats and cholesterol from cells, includingwithin artery wall atheroma, to the liver for excretion orre-utilization.

As used herein, “low-density lipoproteins” refers to a type oflipoprotein that transports fats and cholesterol to cells, and areassociated with increasing rates of accumulation of atherosclerosiswithin the walls of arteries over time.

As used herein, “cholesterol” refers to a type of lipid molecule that isbiosynthesized by all animal cells and is an essential structuralcomponent of the cell membrane. Cholesterol has the following chemicalstructure:

The term “effective amount” means an amount of a compound of the presentinvention that (i) treats or prevents the particular disease, condition,or disorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition, or disorder described herein. In one embodiment, thepresent metabolite or combination of metabolites can be present in aneffective amount for modulating cholesterol, high-density lipoproteins,or low-density lipoproteins in a companion animal.

The dosages expressed herein are in milligrams per kilogram of bodyweight per day (mg/kg/day) unless expressed otherwise.

The term “long-term administration” means periods of repeatedadministration or consumption in excess of one month. Periods of longerthan two, three, or four months can be used for certain embodiments.Also, more extended periods can be used that include longer than 5, 6,7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year can alsobe used. Longer term use extending over 1, 2, 3, or more years areincluded in the invention. For certain aging animals, the animal willcontinue consuming on a regular basis for the remainder of its life.This can also be referred to as consumption for “extended” periods.

The term “regular basis” or “regular administration” means at leastmonthly dosing with the compositions or consumption of the compositions,and in one aspect, means at least weekly dosing. More frequent dosing orconsumption, such as twice or three times weekly, can be performed incertain embodiments. Still, in other embodiments, regimens can be usedthat comprise at least once daily consumption. The skilled artisan willappreciate that the blood level of a compound or certain metabolites ofthat compound or which result after the consumption of that compound,may be a useful tool for assessing or determining dosing frequency. Forexample, for determining feeding amounts for pet food compositionscomprising a certain metabolite, the blood concentration of thatmetabolite, may provide useful information. A frequency, regardless ofwhether expressly exemplified herein, that allows maintenance of adesired blood level of the measured compound, such as a metabolite,within acceptable ranges can be useful herein. The skilled artisan willappreciate that feeding amounts will be a function of the compositionthat is being consumed or administered as well as the animal consumingthe food, and some food compositions may require more or less frequentadministration to maintain a desired blood level of the measuredcompound (e.g., a metabolite).

The relative terms “improve,” “increase,” “enhance,” “decrease” and thelike refer to the effects of the composition disclosed herein (acomposition comprising a metabolites) relative to a composition having alower amount or lacking such metabolites, but otherwise identical.

A “blended” composition merely has at least two components having atleast one different characteristic relative to each other. In oneaspect, moisture content and water activity can be different in thecontext of the present disclosure. In this regard, description of acomposition as “blended” does not imply that the blended composition hasbeen subjected to processing sometimes referenced as “blending,” namelymixing components so that they are indistinguishable from each other,and, in one aspect, such processing is avoided when mixing one componentwith the other components to form a blended composition (e.g., mixing adry component with a wet or semi-moist component). Further in thisregard, in a blended composition each of the at least two componentshaving at least one different characteristic relative to each other canretain their distinct identity and appearance.

“Wet food” means a pet food having a moisture content from about 50% toabout 90%, and in one aspect, from about 70% to about 90%. “Dry food”means a pet food having a moisture content less than about 20%, and inone aspect, less than about 15%, and in a specific aspect, less thanabout 10%. “Semi-moist food” means a pet food having a moisture contentfrom about 20% to about 50%, and in one aspect, from about 25% to about35%.

“Kibbles” is used synonymously with “chunks” herein and both terms meanpieces of dry or semi-moist pet food which can have a pellet shape orany other shape and can be made by slicing a food composition intoseparate pieces. Non-limiting examples of kibbles include particulates;pellets; pieces of pet food, dehydrated meat, meat analog, vegetables,and combinations thereof; and pet snacks, such as meat or vegetablejerky, rawhide, and biscuits. A “meat analog” is a meat emulsion productthat resembles pieces of natural meat in appearance, texture, andphysical structure.

The term “dietary supplement” means a product that is intended to beingested in addition to the normal animal diet. Dietary supplements maybe in any form, e.g., solid, liquid, gel, tablets, capsules, powder, andthe like. In one aspect, they can be provided in convenient dosageforms. In some embodiments, they can be provided in bulk consumerpackages such as bulk powders, liquids, gels, or oils. In otherembodiments, supplements can be provided in bulk quantities to beincluded in other food items such as snacks, treats, supplement bars,beverages and the like.

The compositions disclosed herein may lack any element that is notspecifically disclosed herein. Thus, a disclosure of an embodiment usingthe term “comprising” includes a disclosure of embodiments “consistingessentially of” and “consisting of” the components identified.Similarly, the methods disclosed herein may lack any step that is notspecifically disclosed herein. Thus, a disclosure of an embodiment usingthe term “comprising” includes a disclosure of embodiments “consistingessentially of” and “consisting of” the steps identified. Any embodimentdisclosed herein can be combined with any other embodiment disclosedherein unless explicitly and directly stated otherwise.

The present discussion of embodiments, aspects, examples, etc. areindependent in that they can apply to all methods and compositions. Forexample, a metabolite used in a pet food composition can also be used inthe method of modulating or a method of minimizing costs associated withmaking such a pet food, and vice versa.

Embodiments

In an aspect of the present disclosure, a pet food composition cancomprise protein, carbohydrates, fat, fiber, and a metabolite formodulating at least one of cholesterol, high-density lipoproteins, andlow-density lipoproteins in a companion animal. In one aspect, the petfood composition can include at least 6 distinct metabolites forindividually modulating each one of cholesterol, high-densitylipoproteins, and low-density lipoproteins in a companion animal. In oneembodiment, the pet food composition can provide a decrease in at leastone of cholesterol and low-density lipoproteins in the companion animal.In another embodiment, the pet food composition can provide an increasein high-density lipoproteins in the companion animal. In one aspect, thecompanion animal can be a senior dog or a senior cat.

In another aspect of the present disclosure, a method of modulating atleast one of cholesterol, high-density lipoproteins, or low-densitylipoproteins in a companion animal in a companion animal is provided.The method comprises administering to the companion animal a pet foodcomposition comprising protein, carbohydrates, fat, fiber, and ametabolite for modulating at least one of cholesterol, high-densitylipoproteins, or low-density lipoproteins in the companion animal. Inone aspect, the companion animal can be a senior dog or a senior cat.

Yet another aspect of the present disclosure is a method of minimizingcosts associated with production of a pet food having a firstformulation designed for consumption by a companion animal, such as asenior dog or a senior cat. A further aspect of the present disclosureis a method of enhancing nutritional benefit of a pet food having afirst formulation designed for consumption by a companion animal, suchas a senior dog or a senior cat. These methods comprise adjusting thefirst formulation of the pet food to be a second formulation. At leastone of the first and second formulations comprises a metabolite formodulating at least one of cholesterol, high-density lipoproteins, orlow-density lipoproteins in the companion animal. The adjustingcomprises changing an amount of the metabolite in the first formulationto a different amount in the second formulation.

“Minimizing” costs means that the costs associated with making thesecond formulation are less than the costs associated with making thefirst formulation, for example on a per serving basis, per unit weight,per unit volume, per total energy, and the like. “Enhanced” nutritionalbenefit means that the nutritional benefit of the second formulation isgreater than the nutritional benefit of the first formulation.

As discussed herein, the pet food compositions and methods can contain ametabolite or multiple metabolites for modulating cholesterol,high-density lipoproteins, or low-density lipoproteins. In variousaspects, the compositions and methods can contain 1, 2, 3, 4, 5, 6, 7,8, 9, or even 10 metabolites for any one of the cholesterol,high-density lipoproteins, or low-density lipoproteins, or multipleparameters of cholesterol, high-density lipoproteins, or low-densitylipoproteins, or for each cholesterol, high-density lipoproteins, andlow-density lipoproteins. As such, in one embodiment, the compositioncan comprise at least 12 metabolites, including at least 2 metabolitesfor individually modulating each one of cholesterol, high-densitylipoproteins, and low-density lipoproteins in a companion animal. Inanother embodiment, the composition can comprise at least 18metabolites, including at least 3 metabolites for individuallymodulating each one of cholesterol, high-density lipoproteins, andlow-density lipoproteins in a companion animal. Additionally, in oneembodiment, the composition can comprise at least four distinctmetabolites that modulate at least one of cholesterol, high-densitylipoproteins, or low-density lipoproteins. Further, in one embodiment,the composition can comprise at least five distinct metabolites thatmodulate at least one of cholesterol, high-density lipoproteins, orlow-density lipoproteins. Still, in another embodiment, the method canmodulate at least two parameters selected from the group consisting ofcholesterol, high-density lipoproteins, and low-density lipoproteins andwhere the composition comprises at least six distinct metabolites, threedistinct metabolites for modulating each of the at least two parameters.Yet, in another embodiment, the method can modulate each one ofcholesterol, high-density lipoproteins, and low-density lipoproteins andwhere the composition comprises at least nine distinct metabolites,three distinct metabolites for modulating each of the at least threeparameters. As previously noted, the metabolite andcholesterol/lipoprotein relationships can be used for both method andcomposition embodiments.

In some embodiments, the metabolite negatively modulates the specificcholesterol/lipoprotein, and the changing of the amount of themetabolite comprises decreasing the amount of the metabolite. In otherembodiments, the metabolite negatively modulates the specificcholesterol/lipoprotein, and the changing of the amount of themetabolite comprises increasing the amount of the metabolite. In someembodiments, the metabolite positively modulates the specificcholesterol/lipoprotein, and the changing of the amount of themetabolite comprises increasing the amount of the metabolite. In otherembodiments, the metabolite positively modulates the specificcholesterol/lipoprotein, and the changing of the amount of themetabolite comprises decreasing the amount of the metabolite. These arenot mutually exclusive embodiments; a particular embodiment can comprisedecreasing the amount of a metabolite that negatively modulates a firstspecific cholesterol/lipoprotein and increasing the amount of ametabolite that positively modulates a second specificcholesterol/lipoprotein, and the first and second specificcholesterol/lipoprotein can be the same or different specificcholesterol/lipoprotein (e.g., one or more of cholesterol, high-densitylipoproteins, or low-density lipoproteins).

Decreasing the amount of the metabolite can comprise decreasing theamount of the metabolite directly and/or decreasing the amount of aningredient which comprises the metabolite. In some embodiments,decreasing the amount of the metabolite can comprise decreasing theamount of a precursor of the metabolite directly and/or decreasing theamount of an ingredient which comprises a precursor of the metabolite.For example, the second formulation can contain, relative to the firstformulation, less of an ingredient having a high amount of themetabolite or precursor thereof (e.g., an ingredient having an amount ofthe metabolite or precursor thereof that is higher than in one or moreof the other ingredients).

Increasing the amount of the metabolite can comprise increasing theamount of the metabolite directly and/or increasing the amount of aningredient which comprises the metabolite. In some embodiments,increasing the amount of the metabolite can comprise increasing theamount of a precursor of the metabolite directly and/or increasing theamount of an ingredient which comprises a precursor of the metabolite.For example, the second formulation can contain, relative to the firstformulation, more of an ingredient having a high amount of themetabolite or precursor thereof (e.g., an ingredient having an amount ofthe metabolite or precursor thereof that is higher than in one or moreof the other ingredients).

Generally, the methods and compositions described herein can provide anincrease or a decrease in at least one of cholesterol, high-densitylipoproteins, or low-density lipoproteins in the companion animal. Insome embodiment, the increase or decrease can be at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or even 50%. In one aspect, thecompositions and methods can provide an increase. In another aspect, thecompositions and methods can provide a decrease. In another aspect, thecompositions and methods can provide an increase in one parameter and adecrease in another parameter. As such, the present modulation, andresulting increase or decrease, can provide treatment of one of thefollowing: hypercholesterolemia, hyperlipidemia, coronary heart disease,cerebrovascular disease, peripheral arterial disease, rheumatic heartdisease, deep vein thrombosis, inflammation or pulmonary embolism.

In one embodiment, the increase or decrease can be for at least two ofcholesterol, high-density lipoproteins, and low-density lipoproteins. Inanother embodiment, the increase or decrease can be each one ofcholesterol, high-density lipoproteins, and low-density lipoproteins.

Ingredients comprising the metabolite (e.g., a precursor of themetabolite) and optionally amounts of the metabolite in the ingredientcan be identified by analysis of the ingredient, for example using aseparation technique, such as gas chromatography or liquidchromatography, and then mass spectrometry.

In each of these compositions and methods, the pet food composition canbe a wet food, a semi-moist food or a dry food. In an embodiment, thepet food composition is one or more components of a blended composition.In some embodiments, the pet food composition is a kibble, and in someembodiments, the pet food composition is a meat analog. Additionally, inanother embodiment, the present composition for modulating cholesterol,high-density lipoproteins, and low-density lipoproteins can be a dietarysupplement comprising the metabolites described herein. Further, amethod of modulating cholesterol, high-density lipoproteins, orlow-density lipoproteins can include administering to the companionanimal the dietary supplement.

Such pet food compositions can be administered to the companion animalin amounts ranging from about 3 g of pet food per 1 lb body weight toabout 16 g of pet food per 1 lb body weight of the companion animal.Additionally, the metabolites can be present in amounts from about 0.01weight % to about 10 weight % of the food composition. In one aspect,the metabolites can be present in concentrations of about 0.01 to about1,000 mg/kg of food. In another aspect, the metabolites can be presentin concentrations from about 1 IU to about 500,000 IU per kilogram offood. In one embodiment, the pet food composition can be administered tothe companion animal in amounts sufficient to maintain the health and/orbody weight of the animal. In one aspect, the administration can beregular administration.

As noted above and detailed later in this application, the presentinventors identified metabolite compounds which correlate tocholesterol, high-density lipoproteins, or low-density lipoproteins.Thus, the metabolite in the pet food composition can be one of thesecompounds. Nevertheless, the metabolite can be any metabolite formodulating at least one of cholesterol, high-density lipoproteins, orlow-density lipoproteins in a companion animal, even if the metaboliteis not explicitly disclosed herein. For example, the metabolite can be acompound identified using the methods disclosed herein but not itselfexplicitly disclosed herein. Furthermore, the metabolite can be acompound identified using a method not disclosed herein if the compoundis reliably correlated to at least one of cholesterol, high-densitylipoproteins, or low-density lipoproteins.

As a non-limiting example, the metabolite can modulate cholesterol andcan be selected from the group consisting of palmitoyl sphingomyelin,X-13215, 1-stearoylglycerol (1-monostearin), alpha-tocopherol,7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin),7-beta-hydroxycholesterol, X-17010, glycerophosphorylcholine (GPC),X-16120, 2-linoleoylglycerophosphoethanolamine,5-methyl-2′-deoxycytidine, caproate (6:0),1-linoleoylglycerophosphoethanolamine, glycerol 3-phosphate (G3P),2-arachidonoylglycerophosphoethanolamine, glycerol 2-phosphate,dihydrocholesterol, pelargonate (9:0), stearamide,2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), andmixtures thereof. In one embodiment, the metabolite can modulatecholesterol and can be selected from the group consisting of palmitoylsphingomyelin, 1-stearoylglycerol (1-monostearin), alpha-tocopherol,7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin),7-beta-hydroxycholesterol, glycerophosphorylcholine (GPC),2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine,caproate (6:0), 1-linoleoylglycerophosphoethanolamine, glycerol3-phosphate (G3P), 2-arachidonoylglycerophosphoethanolamine, glycerol2-phosphate, dihydrocholesterol, pelargonate (9:0), stearamide,2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), andmixtures thereof.

As another non-limiting example, the metabolite can modulatehigh-density lipoproteins and can be selected from the group consistingof palmitoyl sphingomyelin, X-13215, alpha-tocopherol,1-stearoylglycerol (1-monostearin), 7-alpha-hydroxycholesterol, X-17010,1-arachidonoylglycerophosphocholine,2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol,1-palmitoylglycerol (1-monopalmitin),1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate(20:4n6), X-17612, 3-hydroxybutyrate (BHBA),2-arachidonoylglycerophosphocholine, pelargonate (9:0), stearamide,N2,N2-dimethylguanosine, 1-linoleoylglycerophosphocholine,dihydrocholesterol, X-11412, 1-stearoylglycerophosphocholine, X-17147,glycerate, heptanoate (7:0), 1-docosahexaenoylglycerophosphocholine,dihomo-linoleate (20:2n6), 2-linoleoylglycerophosphocholine,2-palmitoylglycerophosphocholine, and mixtures thereof. In oneembodiment, the metabolite can modulate high-density lipoproteins andcan be selected from the group consisting of palmitoyl sphingomyelin,alpha-tocopherol, 1-stearoylglycerol (1-monostearin),7-alpha-hydroxycholesterol, 1-arachidonoylglycerophospho choline,2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol,1-palmitoylglycerol (1-monopalmitin),1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate(20:4n6), 3-hydroxybutyrate (BHBA), 2-arachidonoylglycerophosphocholine,pelargonate (9:0), stearamide, N2,N2-dimethylguanosine,1-linoleoylglycerophosphocholine, dihydrocholesterol,1-stearoylglycerophosphocholine, glycerate, heptanoate (7:0),1-docosahexaenoylglycerophosphocholine, dihomo-linoleate (20:2n6),2-linoleoylglycerophosphocholine, 2-palmitoylglycerophosphocholine, andmixtures thereof.

As yet another non-limiting example, the metabolite can modulatelow-density lipoproteins and can be selected from the group consistingof 1-stearoylglycerol (1-monostearin), X-13215, palmitoyl sphingomyelin,alpha-tocopherol, 7-alpha-hydroxycholesterol,2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC),7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine,X-17010, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol(1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine,2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate,glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine,N2,N2-dimethylguanosine, and mixtures thereof. In one embodiment, themetabolite can modulate low-density lipoproteins and can be selectedfrom the group consisting of 1-stearoylglycerol (1-monostearin),palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol,2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC),7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine,2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol(1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine,2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate,glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine,N2,N2-dimethylguanosine, and mixtures thereof.

In yet another aspect of the present disclosure, a method enhancesnutritional benefit of a pet food having a first formulation designedfor consumption by companion animals, and the method comprisesadministering the pet food having the first formulation to a firstcompanion animal. The method further comprises measuring in a sample ofbody fluid from the companion animal (e.g., plasma) an amount of asurrogate marker comprising a metabolite that modulates at least one ofcholesterol, high-density lipoproteins, or low-density lipoproteins. Themethod further comprises adjusting the first formulation of the pet foodto be a second formulation having a difference from the firstformulation selected from the group consisting of (i) an ingredient ispresent in the second formulation and is absent in the firstformulation, (ii) an ingredient is absent in the second formulation andis present in the first formulation, (iii) an ingredient is present inthe first and second formulations but in a different amount, and (iv)combinations thereof. The adjusting is based at least partially on theamount of the surrogate marker measured in the previous step.

The adjusting can comprise directly decreasing the amount of ametabolite negatively modulating a specific cholesterol/lipoproteinand/or decreasing the amount of an ingredient which comprises ametabolite negatively modulating a specific cholesterol/lipoprotein. Insome embodiments, decreasing the amount of the metabolite can comprisedecreasing the amount of a precursor of the metabolite directly and/ordecreasing the amount of an ingredient which comprises a precursor ofthe metabolite. For example, the second formulation can contain,relative to the first formulation, less of an ingredient having a highamount of the metabolite or precursor thereof (e.g., an ingredienthaving an amount of the metabolite or precursor thereof that is higherthan in one or more of the other ingredients).

The adjusting can comprise increasing the amount of the metabolite cancomprise directly increasing the amount of a metabolite positivelymodulating a specific cholesterol/lipoprotein and/or increasing theamount of an ingredient which comprises a metabolite positivelymodulating a specific cholesterol/lipoprotein. In some embodiments,increasing the amount of the metabolite can comprise increasing theamount of a precursor of the metabolite directly and/or increasing theamount of an ingredient which comprises a precursor of the metabolite.For example, the second formulation can contain, relative to the firstformulation, more of an ingredient having a high amount of themetabolite or precursor thereof (e.g., an ingredient having an amount ofthe metabolite or precursor thereof that is higher than in one or moreof the other ingredients).

As noted above, ingredients comprising the metabolite (e.g., a precursorof the metabolite) and optionally amounts of the metabolite in theingredient can be identified by analysis of the ingredient, for exampleusing a separation technique, such as gas chromatography or liquidchromatography, and then mass spectrometry.

The method further comprises producing the pet food in the secondformulation. In an embodiment, the method comprises administering thepet food having the second formulation to a second companion animal.

This method can be used to provide customized nutrition for a specificcompanion animal. For example, the first and second companion animal canbe the same specific companion animal such that the animal who isadministered the pet food having the first formulation has one or moreof their specific cholesterol/lipoprotein of the first formulationassessed. Then this same animal is provided with the resultant secondformulation which this animal will more effectively modulate withrespect to at least one of cholesterol, high-density lipoproteins, orlow-density lipoproteins. Consequently, a pet owner can compensate fortheir pet's age-induced changes in one or more of cholesterol,high-density lipoproteins, or low-density lipoproteins.

Alternatively or additionally, this method can be used to providecustomized nutrition for companion animals who share one or more of agender, an approximate age, an approximate size (e.g., body weight,height, and/or length) or a breed. For example, the second companionanimal can be a different specific animal than the first companionanimal but has a characteristic selected from the group consisting of(i) about the same age as the first companion animal, (ii) about thesame size as the first companion animal, (iii) the same breed as thefirst companion animal, (iv) the same gender as the first companionanimal, and (iv) combinations thereof. In one embodiment, the secondcompanion animal can be one of a plurality of companion animals who eachshare the characteristic with the first companion animal. The method cancomprise administering the pet food having the second formulation to theplurality of companion animals. In an embodiment, at least a portion ofthe plurality of companion animals is located remotely relative to thefirst companion animal.

The pet food compositions disclosed herein can be any food formulatedfor consumption by a pet such as a companion animal. In an embodiment,the pet food composition provides complete nutrition as defined by theAssociation of American Feed Control Officials (AAFCO) and which dependson the type of animal for which the composition is intended (e.g., dogor cat).

The pet food composition can comprise meat, such as emulsified meat.Examples of suitable meat include poultry, beef, pork, lamb and fish,especially those types of meats suitable for pets. The meat can includeany additional parts of an animal including offal. Some or all of themeat can be provided as one or more meat meals, namely meat that hasbeen dried and ground to form substantially uniform-sized particles andas defined by AAFCO. Additionally or alternatively, vegetable proteincan be used, such as pea protein, corn protein (e.g., ground corn orcorn gluten), wheat protein (e.g., ground wheat or wheat gluten), soyprotein (e.g., soybean meal, soy concentrate, or soy isolate), riceprotein (e.g., ground rice or rice gluten) and the like.

The pet food compositions disclosed herein can comprise vegetable oil, aflavorant, a colorant and water. Suitable vegetable oils include soybeanoil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil,safflower oil, and the like. Examples of suitable flavorants includeyeast, tallow, rendered animal meals (e.g., poultry, beef, lamb, pork),flavor extracts or blends (e.g., grilled beef), animal digests, and thelike. Suitable colorants include FD&C colors, such as blue no. 1, blueno. 2, green no. 3, red no. 3, red no. 40, yellow no. 5, yellow no. 6,and the like; natural colors, such as caramel coloring, annatto,chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene,elderberry juice, pandan, butterfly pea and the like; titanium dioxide;and any suitable food colorant known to the skilled artisan.

The pet food compositions disclosed herein can optionally includeadditional ingredients, such as other grains and/or other starchesadditionally or alternatively to flour, amino acids, fibers, sugars,animal oils, aromas, other oils additionally or alternatively tovegetable oil, humectants, preservatives, polyols, salts, oral careingredients, antioxidants, vitamins, minerals, probiotic microorganisms,bioactive molecules or combinations thereof.

Suitable starches include a grain such as corn, rice, wheat, barley,oats, soy and the like, and mixtures of these grains, and can beincluded at least partially in any flour. Suitable humectants includesalt, sugars, propylene glycol and polyhydric glycols such as glycerinand sorbitol, and the like. Suitable oral care ingredients includealfalfa nutrient concentrate containing chlorophyll, sodium bicarbonate,phosphates (e.g., tricalcium phosphate, acid pyrophosphates, tetrasodiumpyrophosphate, metaphosphates, and orthophosphates), peppermint, cloves,parsley, ginger and the like. Examples of suitable antioxidants includebutylated hydroxyanisole (“BHA”) and butylated hydroxytoluene (“BHT”),vitamin E (tocopherols), and the like.

Non-limiting examples of vitamins that can be used include Vitamins A,B-complex (such as B-1, B-2, B-6 and B-12), C, D, E and K, niacin andacid vitamins such as pantothenic acid and folic acid and biotin.Non-limiting examples of suitable minerals include calcium, iron, zinc,magnesium, iodine, copper, phosphorus, manganese, potassium, chromium,molybdenum, selenium, nickel, tin, silicon, vanadium, boron and thelike.

-   -   n-limiting examples of suitable preservatives include potassium        sorbate, sorbic acid, sodium methyl para-hydroxybenzoate,        calcium propionate, propionic acid, and combinations thereof.

Specific amounts for each additional ingredient in the pet foodcompositions disclosed herein will depend on a variety of factors suchas the ingredient included in the first edible material and any secondedible material; the species of animal; the animal's age, body weight,general health, sex, and diet; the animal's consumption rate; thepurpose for which the food product is administered to the animal; andthe like. Therefore, the components and their amounts may vary widely.

For example, the amount of any of the above-noted ingredients can bedecreased or increased based on the estimated effect on one or more ofcholesterol, high-density lipoproteins, or low-density lipoproteins(e.g., an effect identified by one of the methods disclosed herein). Inan embodiment, the amount of one or more of the above-noted ingredientscan be increased if such ingredients comprise a metabolite thatpositively modulates one or more of cholesterol, high-densitylipoproteins, or low-density lipoproteins. Additionally oralternatively, the amount of one or more of the above-noted ingredientscan be decreased if such ingredients comprise a metabolite thatnegatively modulates one or more of cholesterol, high-densitylipoproteins, or low-density lipoproteins.

As noted above, ingredients comprising the metabolite (e.g., a precursorof the metabolite) and optionally amounts of the metabolite in theingredient can be identified by analysis of the ingredient, for exampleusing a separation technique, such as gas chromatography or liquidchromatography, and then mass spectrometry.

EXAMPLES

The following non-limiting examples are illustrative of embodiments ofthe present disclosure.

Methods

Each of the examples was derived from the following study.

83 Canines were all fed Diet A for 5 weeks, followed by a 1 weektransition period and then 15 were fed Diet B for 5 weeks as shown inthe Table 1 below. Plasma samples were taken after overnight fastingusing EDTA vacutainer tubes during the fifth week of feeding of eachdiet. After centrifugation, plasma was aliquoted into cryovials andfrozen at −80° C. Cholesterol, high-density lipoproteins, andlow-density lipoproteins were measured using the Cobas® c311 or e411clinical chemistry analyzer, according to manufacturer's directions.

TABLE 1 Moisture Basis Moisture % DM % Protein % Fat % Ash % Fiber % CHO% GE kcal/g Diet A As-Is 8.1 91.9 22.7 13.3 6.1 2.0 47.9 4.5 Dry 0 10024.7 14.5 6.6 2.1 52.1 4.9 matter Diet B As-Is 76 24 9.1 10.5 1.8 0 2.61.7 Dry 0 100 38 43.7 7.5 0 10.8 6.9 Matter

Metabolomic analysis was carried out using the following methods byMetabolon Inc. Samples were extracted and split into equal parts foranalysis on GC/MS and LC/MS/MS platforms. Proprietary software was usedto match ions to an in-house library of standards for metaboliteidentification and for metabolite quantitation by peak area integrationby Metabolon Inc. Mass and retention index are provided in the followingtables such that each metabolite can be uniquely identified andindividually distinguished.

At the time of analysis, samples were thawed and extracts prepared toremove protein, dislodge small molecules bound to protein or physicallytrapped in the precipitated protein matrix, and recover a wide range ofchemically diverse metabolites. A separate aliquot of each experimentalplasma sample was taken then pooled for the creation of “Client Matrix”(CMTRX) samples. These CMTRX samples were injected throughout theplatform run and served as technical replicates, allowing variability inthe quantitation of all consistently detected biochemicals to bedetermined and overall process variability and platform performance tobe monitored. Extracts of all experimental and CMTRX samples were splitfor analysis on the GC/MS and LC/MS/MS platforms.

The CMTRX technical replicate samples were treated independentlythroughout the process as if they were client study samples. All processsamples (CMTRX and Grob test mixtures of organic components used toassess GC column performance, process blanks, etc.) were spaced evenlyamong the injections for each day and all client samples were randomlydistributed throughout each day's run. Data were collected over multipleplatform run days and thus ‘block normalized’ by calculating the medianvalues for each run-day block for each individual compound. Thisnormalization minimizes any inter-day instrument gain or drift, but doesnot interfere with intra-day sample variability. Missing values (if any)were assumed to be below the level of detection for that biochemicalwith the instrumentation used and were imputed with the observed minimumfor that particular biochemical.

A number of internal standards were added to each experimental andprocess standard sample just prior to injection into the massspectrometers. A measure of the platform variability (7%) was determinedby calculating the median relative standard deviation (RSD) for theseinternal standards. Because these standards are added to the samplesimmediately prior to injection into the instrument, this value reflectsinstrument variation. In addition, the median relative standarddeviation (RSD) for the biochemicals that were consistently measured inthe CMTRX represents the total variability within the process for theactual experimental samples and the variability in quantitation of theendogenous metabolites within these samples (12%). Results for the CMTRXand internal standards indicated that the platform produced data thatmet process specifications.

589 total metabolites were detected in plasma. This total corresponds tomany biochemicals (401) that matched a named structure in the referencelibrary (named compounds). The remaining biochemicals (188) representdistinct chemical entities (that is, they represent a single molecule ofdiscrete molecular formula and structure), but they do not currentlymatch a named biochemical in the reference library (unnamed/unknowncompounds).

Example 1 (Cholesterol)

Metabolite correlations with cholesterol were determined based on plasmametabolomics (Table 2). This provided a predictive model of compoundswhich can influence cholesterol either positively or negatively. Feedingdifferent levels of these compounds (diet B vs diet A; Table 3), andnoting changes in cholesterol (Table 4), served as a validation model.The metabolite compositions of the two different diets were determinedto identify relative levels of specific compounds. Those validated bythe model are shown in Table 5.

TABLE 2 Metabolite correlations with cholesterol. Correlations with a Pvalue < 0.01 are reported. Correlation Retention ID Correlation P-ValueIndex Mass palmitoyl sphingomyelin 0.7526 0.00E+00 2524 311.3 X - 132150.7468 0.00E+00 5179 149.2 1-stearoylglycerol (1-monostearin) 0.70031.11E−15 2186.6 399.4 alpha-tocopherol 0.6558 2.32E−13 2305.4 502.5stearoyl sphingomyelin 0.6081 3.13E−11 #N/A #N/A7-alpha-hydroxycholesterol 0.523 3.29E−08 2300 456.2 2-ethylhexanoate0.4816 5.17E−07 1210.5 201.2 1-palmitoylglycerol (1-monopalmitin) 0.46071.81E−06 2119.5 371.3 dimethylarginine (SDMA + ADMA) −0.455 2.51E−06 812203.2 7-beta-hydroxycholesterol 0.4381 6.43E−06 2340 456.4 X - 170100.4193 1.73E−05 3169.5 189 glycerophosphorylcholine (GPC) 0.41342.33E−05 694 258.1 X - 16120 0.4021 4.06E−05 1271.7 164.1 lathosterol0.3998 4.53E−05 2337 255.2 2-linoleoylglycerophosphoethanolamine 0.37680.0001 5650 476.4 S-propylcysteine −0.3822 0.0001 2000.5 164.15-methyl-2′-deoxycytidine −0.3627 0.0002 1480 242 caproate (6:0) 0.36780.0002 2766 115.2 X - 11987 −0.3694 0.0002 1409 139.2 threonine 0.36110.0003 713 120.1 lysine 0.3529 0.0004 1836.7 317.2 X - 12355 −0.3490.0004 3559 206.1 1-linoleoylglycerophosphoethanolamine 0.3451 0.00055725 476.3 threonate 0.337 0.0007 1560.7 292.1 glycerol 3-phosphate(G3P) 0.3318 0.0008 1719.7 357.1 arabinose 0.3229 0.0012 1631.6 217N2,N2-dimethylguanosine −0.3207 0.0013 2157 312.12-arachidonoylglycerophosphoethanolamine 0.3084 0.002 5674 500.3glycerol 2-phosphate 0.3047 0.0023 1691.8 243 dihydrocholesterol 0.30330.0024 2320.8 215.2 pelargonate (9:0) 0.3008 0.0026 4847 157.2 caprylate(8:0) 0.298 0.0029 4367 143.2 stearamide 0.2949 0.0032 2071 1312-palmitoylglycerophosphoethanolamine 0.294 0.0033 5790 452.3 X - 11437−0.2938 0.0033 2888 231 X - 16940 −0.2931 0.0034 1694.1 204.9 X - 120290.2834 0.0047 683 329.1 N1-methyladenosine −0.2791 0.0054 #N/A #N/Aalpha-ketoglutarate −0.276 0.0059 1779 419.1 4-vinylphenol sulfate−0.2711 0.0069 3323 199.1 X - 16945 −0.2702 0.0071 3457.9 351 thymolsulfate −0.2687 0.0075 4395 229.1 malate −0.2634 0.0088 1502 233pyrophosphate (PPi) −0.2629 0.0089 1642.3 451 cystine 0.2605 0.00962015.3 145.9 heptanoate (7:0) 0.2599 0.0097 3695 129.2 X - 12668 −0.25990.0098 2318 246.1

TABLE 3 Compound levels present in diets A and B. Values representscaled imputed values (data are scaled such that the median valuemeasured across all samples was set to 1.0 and missing values (if any)were imputed with the observed minimum for that particular compound). IDA B palmitoyl sphingomyelin 0.03 0.2 X - 13215 0.02 0.11-stearoylglycerol (1-monostearin) 0.38 1.21 alpha-tocopherol 0.01 0.03stearoyl sphingomyelin N/A N/A 7-alpha-hydroxycholesterol 0.74 4.912-ethylhexanoate 0.06 0.02 1-palmitoylglycerol (1-monopalmitin) 0.871.52 dimethylarginine (SDMA + ADMA) 0.29 0.63 7-beta-hydroxycholesterol0.91 1.81 X - 17010 0.68 2.51 glycerophosphorylcholine (GPC) 57.82106.56 X - 16120 0.45 0.45 lathosterol 0.41 0.412-linoleoylglycerophosphoethanolamine 0.56 2.58 S-propylcysteine 0.050.05 5-methyl-2′-deoxycytidine 1.05 0.61 caproate (6:0) 0.35 0.49 X -11987 0.14 0.14 threonine 3.43 3.44 lysine 33.69 6.43 X - 12355 0.030.03 1-linoleoylglycerophosphoethanolamine 0.48 2.77 threonate 0.61 0.22glycerol 3-phosphate (G3P) 3.12 10.24 arabinose 10.21 2.48N2,N2-dimethylguanosine 0.59 0.542-arachidonoylglycerophosphoethanolamine 0.33 10.33 glycerol 2-phosphate3.63 4.01 dihydrocholesterol 0.23 0.74 pelargonate (9:0) 0.07 0.1caprylate (8:0) 0.1 0.12 stearamide 0.99 1.062-palmitoylglycerophosphoethanolamine 0.69 2.75 X - 11437 0.05 0.05 X -16940 0.12 0.12 X - 12029 0.35 0.35 N1-methyladenosine #N/A #N/Aalpha-ketoglutarate 0.22 0.43 4-vinylphenol sulfate 0.02 0.02 X - 169450.07 0.07 thymol sulfate 0.41 0.41 malate 9.52 4.85 pyrophosphate (PPi)0.15 0.34 cystine 0.37 0.37 heptanoate (7:0) 0.27 0.39 X - 12668 0.130.13

TABLE 4 Cholesterol in response to diet A and Diet B. Difference Diet ADiet B p- Parameter Mean StdDev Mean StdDev Mean StdErr valueCholesterol 190 36.0 253.4 38.7 −63.4 5.2 0

TABLE 5 Compounds validated by dietary change. Retention ID Index Masspalmitoyl sphingomyelin 2524 311.3 X - 13215 5179 149.21-stearoylglycerol (1-monostearin) 2186.6 399.4 alpha-tocopherol 2305.4502.5 7-alpha-hydroxycholesterol 2300 456.2 1-palmitoylglycerol(1-monopalmitin) 2119.5 371.3 7-beta-hydroxycholesterol 2340 456.4 X -17010 3169.5 189 glycerophosphorylcholine (GPC) 694 258.1 X - 161201271.7 164.1 2-linoleoylglycerophosphoethanolamine 5650 476.45-methyl-2′-deoxycytidine 1480 242 caproate (6:0) 2766 115.21-linoleoylglycerophosphoethanolamine 5725 476.3 glycerol 3-phosphate(G3P) 1719.7 357.1 2-arachidonoylglycerophosphoethanolamine 5674 500.3glycerol 2-phosphate 1691.8 243 dihydrocholesterol 2320.8 215.2pelargonate (9:0) 4847 157.2 stearamide 2071 1312-palmitoylglycerophosphoethanolamine 5790 452.3 malate 1502 233heptanoate (7:0) 3695 129.2

Example 2 (High-Density Lipoprotein)

Metabolite correlations with high-density lipoproteins were determinedbased on plasma metabolomics (Table 6). This provided a predictive modelof compounds which can influence high-density lipoproteins eitherpositively or negatively. Feeding different levels of these compounds(diet B vs diet A; Table 7), and noting changes in high-densitylipoproteins (Table 8), served as a validation model. The metabolitecompositions of the two different diets were determined to identifyrelative levels of specific compounds. Those compounds validated by themodel are shown in Table 9).

TABLE 6 Metabolite correlations with high-density lipoproteins.Correlations with a P value < 0.01 are reported. Correlation RetentionID Correlation P-Value Index Mass palmitoyl sphingomyelin 0.75780.00E+00 2524 311.3 stearoyl sphingomyelin 0.7318 0.00E+00 N/A N/A X -13215 0.6493 4.78E−13 5179 149.2 alpha-tocopherol 0.6197 1.03E−11 2305.4502.5 1-stearoylglycerol (1-monostearin) 0.5654 1.32E−09 2186.6 399.42-ethylhexanoate 0.4585 2.06E−06 1210.5 201.2 X - 16120 0.4562 2.35E−061271.7 164.1 7-alpha-hydroxycholesterol 0.4552 2.48E−06 2300 456.2 X -17010 0.4338 8.11E−06 3169.5 189 dimethylarginine (SDMA + ADMA) −0.43039.76E−06 812 203.2 threonate 0.4123 2.45E−05 1560.7 292.1S-propylcysteine −0.4033 3.83E−05 2000.5 164.1 X - 12355 −0.39146.75E−05 3559 206.1 1-arachidonoylglycerophosphocholine 0.375 0.00015554 544.3 2-docosapentaenoylglycerophosphocholine 0.3744 0.0001 5532570.3 X - 11987 −0.3817 0.0001 1409 139.2 7-beta-hydroxycholesterol0.3683 0.0002 2340 456.4 1-palmitoylglycerol (1-monopalmitin) 0.35980.0003 2119.5 371.3 X - 16940 −0.3518 0.0004 1694.1 204.9 caprylate(8:0) 0.3467 0.0005 4367 143.2 X - 11549 0.3379 0.0007 5093 339.31-docosapentaenoylglycerophosphocholine 0.3357 0.0007 5584 570.4caproate (6:0) 0.3355 0.0007 2766 115.2 arachidonate (20:4n6) 0.33450.0008 5525 303.4 X - 17612 0.3344 0.0008 4298.5 567.4 3-hydroxybutyrate(BHBA) 0.3321 0.0008 1203.5 116.9 2-arachidonoylglycerophosphocholine0.3236 0.0012 5524 544.3 2-stearoylglycerophosphocholine 0.3234 0.00125780 524.4 pelargonate (9:0) 0.3229 0.0012 4847 157.2 X - 15546 −0.31850.0014 1861 271.1 N-stearoyl taurine 0.3169 0.0015 5785.9 390.3glutarylcarnitine (C5) 0.3149 0.0016 1565 276.1 benzoate 0.3109 0.00181291.5 179 threonine 0.3086 0.002 713 120.1 stearamide 0.3069 0.00212071 131 X - 16009 0.3068 0.0021 2191 222 N2,N2-dimethylguanosine−0.3065 0.0021 2157 312.1 1-linoleoylglycerophosphocholine 0.3036 0.00245574 520.4 dihydrocholesterol 0.2989 0.0028 2320.8 215.2 X - 114120.2977 0.0029 3836 204.2 1-stearoylglycerophosphocholine 0.2934 0.00345844 524.4 X - 17147 0.2882 0.004 4830 187.2 glycerol 0.2845 0.0045 1311205 X - 12435 0.2824 0.0048 3174 357.2 glycerate 0.2818 0.0049 1360.7189 cysteine-glutathione disulfide −0.2804 0.0052 821 427.1 heptanoate(7:0) 0.2792 0.0054 3695 129.2 X - 16945 −0.2783 0.0055 3457.9 3514-vinylphenol sulfate −0.2775 0.0057 3323 199.11-docosahexaenoylglycerophosphocholine 0.277 0.0058 5518 568.4N-palmitoyl taurine 0.2756 0.006 5556.8 362.4 dihomo-linoleate (20:2n6)0.2755 0.006 5722 307.3 succinylcarnitine 0.2754 0.0061 1401 262.12-linoleoylglycerophosphocholine 0.2717 0.0068 5544 520.4 X - 13543−0.2669 0.0079 1096 484.1 X - 11437 −0.2656 0.0082 2888 2312-palmitoylglycerophosphocholine 0.2592 0.0099 5604 496.3

TABLE 7 Compound levels present in diets A and B. Values representscaled imputed values (data are scaled such that the median valuemeasured across all samples was set to 1.0 and missing values (if any)were imputed with the observed minimum for that particular compound). IDA B palmitoyl sphingomyelin 0.03 0.2 stearoyl sphingomyelin N/A N/A X -13215 0.02 0.1 alpha-tocopherol 0.01 0.03 1-stearoylglycerol(1-monostearin) 0.38 1.21 2-ethylhexanoate 0.06 0.02 X - 16120 0.45 0.457-alpha-hydroxycholesterol 0.74 4.91 X - 17010 0.68 2.51dimethylarginine (SDMA + ADMA) 0.29 0.63 threonate 0.61 0.22S-propylcysteine 0.05 0.05 X - 12355 0.03 0.031-arachidonoylglycerophosphocholine 0.03 0.722-docosapentaenoylglycerophosphocholine 0.11 1.23 X - 11987 0.14 0.147-beta-hydroxycholesterol 0.91 1.81 1-palmitoylglycerol (1-monopalmitin)0.87 1.52 X - 16940 0.12 0.12 caprylate (8:0) 0.1 0.12 X - 11549 0.130.13 1-docosapentaenoylglycerophosphocholine 0.22 1.12 caproate (6:0)0.35 0.49 arachidonate (20:4n6) 0.43 4.48 X - 17612 0.21 0.33-hydroxybutyrate (BHBA) 0.18 0.34 2-arachidonoylglycerophosphocholine0.05 1.4 2-stearoylglycerophosphocholine 0.02 0.2 pelargonate (9:0) 0.070.1 X - 15546 0.15 0.15 N-stearoyl taurine 2.12 1.18 glutarylcarnitine(C5) 0.27 0.27 benzoate 0.03 0.03 threonine 3.43 3.44 stearamide 0.991.06 X - 16009 0.2 0.2 N2,N2-dimethylguanosine 0.59 0.541-linoleoylglycerophosphocholine 0.31 0.93 dihydrocholesterol 0.23 0.74X - 11412 0.13 0.21 1-stearoylglycerophosphocholine 0.01 0.45 X - 171470.07 0.08 glycerol 2.61 1.85 X - 12435 0.36 0.36 glycerate 3.38 9.94cysteine-glutathione disulfide 0.04 0.04 heptanoate (7:0) 0.27 0.39 X -16945 0.07 0.07 4-vinylphenol sulfate 0.02 0.021-docosahexaenoylglycerophosphocholine 0.07 0.88 N-palmitoyl taurine 10.94 dihomo-linoleate (20:2n6) 0.33 4.06 succinylcarnitine 0.32 0.322-linoleoylglycerophosphocholine 0.2 1.75 X - 13543 0.1 0.1 X - 114370.05 0.05 2-palmitoylglycerophosphocholine 0.08 0.9

TABLE 8 High-density lipoproteins in response to diet A and Diet B.Difference Diet A Diet B p- Parameter Mean StdDev Mean StdDev MeanStdErr value High-Density 170.7 28.9 209.6 24.4 −38.9 4.8 0 Lipoprotein

TABLE 9 Compounds validated by dietary change. Retention ID Index Masspalmitoyl sphingomyelin 2524 311.3 X - 13215 5179 149.2 alpha-tocopherol2305.4 502.5 1-stearoylglycerol (1-monostearin) 2186.6 399.47-alpha-hydroxycholesterol 2300 456.2 X - 17010 3169.5 1891-arachidonoylglycerophosphocholine 5554 544.32-docosapentaenoylglycerophosphocholine 5532 570.37-beta-hydroxycholesterol 2340 456.4 1-palmitoylglycerol(1-monopalmitin) 2119.5 371.3 1-docosapentaenoylglycerophosphocholine5584 570.4 caproate (6:0) 2766 115.2 arachidonate (20:4n6) 5525 303.4X - 17612 4298.5 567.4 3-hydroxybutyrate (BHBA) 1203.5 116.92-arachidonoylglycerophosphocholine 5524 544.3 pelargonate (9:0) 4847157.2 stearamide 2071 131 N2,N2-dimethylguanosine 2157 312.11-linoleoylglycerophosphocholine 5574 520.4 dihydrocholesterol 2320.8215.2 X - 11412 3836 204.2 1-stearoylglycerophosphocholine 5844 524.4X - 17147 4830 187.2 glycerate 1360.7 189 heptanoate (7:0) 3695 129.21-docosahexaenoylglycerophosphocholine 5518 568.4 dihomo-linoleate(20:2n6) 5722 307.3 2-linoleoylglycerophosphocholine 5544 520.42-palmitoylglycerophosphocholine 5604 496.3

Example 3 (Low-Density Lipoprotein)

Metabolite correlations with low-density lipoproteins were determinedbased on plasma metabolomics (Table 10). This provided a predictivemodel of compounds which can influence low-density lipoproteins eitherpositively or negatively. Feeding different levels of these compounds(diet B vs diet A; Table 11), and noting changes in low-densitylipoproteins (Table 12), served as a validation model. The metabolitecompositions of the two different diets were determined in order toidentify relative levels of specific compounds. Those compoundsvalidated by the model are shown in Table 13).

TABLE 10 Metabolite correlations with low-density lipoproteins.Correlations with a P value < 0.01 are reported. Correlation RetentionID Correlation P-Value Index Mass 1-stearoylglycerol (1-monostearin)0.5795 4.04E−10 2186.6 399.4 X - 13215 0.5664 1.21E−09 5179 149.2palmitoyl sphingomyelin 0.5397 9.79E−09 2524 311.3 alpha-tocopherol0.4864 3.82E−07 2305.4 502.5 lysine 0.4624 1.64E−06 1836.7 317.2lathosterol 0.4583 2.08E−06 2337 255.2 7-alpha-hydroxycholesterol 0.4376.85E−06 2300 456.2 arabinose 0.4178 1.86E−05 1631.6 2172-linoleoylglycerophosphoethanolamine 0.4106 2.68E−05 5650 476.4glycerophosphorylcholine (GPC) 0.4041 3.68E−05 694 258.17-beta-hydroxycholesterol 0.4028 3.93E−05 2340 456.4 dimethylarginine(SDMA + ADMA) −0.3911 6.83E−05 812 203.21-linoleoylglycerophosphoethanolamine 0.3485 0.0004 5725 476.3 X - 120290.3474 0.0005 683 329.1 X - 12189 0.3425 0.0006 1249 273.1 X - 170100.334 0.0008 3169.5 189 2-palmitoylglycerophosphoethanolamine 0.33150.0009 5790 452.3 stearoyl sphingomyelin 0.327 0.001 #N/A #N/A1-palmitoylglycerol (1-monopalmitin) 0.3154 0.0016 2119.5 371.3 caproate(6:0) 0.3147 0.0016 2766 115.2 N1-methyladenosine −0.3115 0.0018 #N/A#N/A 2-ethylhexanoate 0.3045 0.0023 1210.5 201.2 cystine 0.3045 0.00232015.3 145.9 X - 11987 −0.2896 0.0038 1409 139.25-methyl-2′-deoxycytidine −0.2866 0.0042 1480 242 threonine 0.284 0.0046713 120.1 2-arachidonoylglycerophosphoethanolamine 0.2796 0.0053 5674500.3 S-propylcysteine −0.2788 0.0054 2000.5 164.1 malate −0.2732 0.00651502 233 taurocholate 0.272 0.0067 5122 514.3 glycerol 2-phosphate0.2683 0.0076 1691.8 243 glycerol 3-phosphate (G3P) 0.2648 0.0084 1719.7357.1 X - 12355 −0.2621 0.0091 3559 206.12-oleoylglycerophosphoethanolamine 0.2601 0.0097 5848 478.3N2,N2-dimethylguanosine −0.2598 0.0098 2157 312.1

TABLE 11 Compound levels present in diets A and B. Values representscaled imputed values (data are scaled such that the median valuemeasured across all samples was set to 1.0 and missing values (if anywere imputed with the observed minimum for that particular compound). IDA B 1-stearoylglycerol (1-monostearin) 0.38 1.21 X - 13215 0.02 0.1palmitoyl sphingomyelin 0.03 0.2 alpha-tocopherol 0.01 0.03 lysine 33.696.43 lathosterol 0.41 0.41 7-alpha-hydroxycholesterol 0.74 4.91arabinose 10.21 2.48 2-linoleoylglycerophosphoethanolamine 0.56 2.58glycerophosphorylcholine (GPC) 57.82 106.56 7-beta-hydroxycholesterol0.91 1.81 dimethylarginine (SDMA + ADMA) 0.29 0.631-linoleoylglycerophosphoethanolamine 0.48 2.77 X - 12029 0.35 0.35 X -12189 0.23 0.23 X - 17010 0.68 2.512-palmitoylglycerophosphoethanolamine 0.69 2.75 stearoyl sphingomyelinN/A N/A 1-palmitoylglycerol (1-monopalmitin) 0.87 1.52 caproate (6:0)0.35 0.49 N1-methyladenosine N/A N/A 2-ethylhexanoate 0.06 0.02 cystine0.37 0.37 X - 11987 0.14 0.14 5-methyl-2′-deoxycytidine 1.05 0.61threonine 3.43 3.44 2-arachidonoylglycerophosphoethanolamine 0.33 10.33S-propylcysteine 0.05 0.05 malate 9.52 4.85 taurocholate 6.67 0.61glycerol 2-phosphate 3.63 4.01 glycerol 3-phosphate (G3P) 3.12 10.24 X -12355 0.03 0.03 2-oleoylglycerophosphoethanolamine 0.43 6.03N2,N2-dimethylguanosine 0.59 0.54

TABLE 12 Low-density lipoproteins in response to diet A and Diet B.Difference Diet A Diet B p- Parameter Mean StdDev Mean StdDev MeanStdErr value Low-Density 8.3 4.7 31.5 18.2 −23.2 3.6 0 Lipoproteins

TABLE 13 Compounds validated by dietary change. Retention ID Index Mass1-stearoylglycerol (1-monostearin) 2186.6 399.4 X - 13215 5179 149.2palmitoyl sphingomyelin 2524 311.3 alpha-tocopherol 2305.4 502.57-alpha-hydroxycholesterol 2300 456.22-linoleoylglycerophosphoethanolamine 5650 476.4glycerophosphorylcholine (GPC) 694 258.1 7-beta-hydroxycholesterol 2340456.4 1-linoleoylglycerophosphoethanolamine 5725 476.3 X - 17010 3169.5189 2-palmitoylglycerophosphoethanolamine 5790 452.3 1-palmitoylglycerol(1-monopalmitin) 2119.5 371.3 caproate (6:0) 2766 115.25-methyl-2′-deoxycytidine 1480 2422-arachidonoylglycerophosphoethanolamine 5674 500.3 malate 1502 233glycerol 2-phosphate 1691.8 243 glycerol 3-phosphate (G3P) 1719.7 357.12-oleoylglycerophosphoethanolamine 5848 478.3 N2,N2-dimethylguanosine2157 312.1

It should be understood that various changes and modifications to thepresently embodiments described herein will be apparent to those skilledin the art. Such changes and modifications can be made without departingfrom the spirit and scope of the present subject matter and withoutdiminishing its intended advantages. It is therefore intended that suchchanges and modifications be covered by the appended claims.

The invention is claimed as follows:
 1. A pet food composition,comprising: protein, carbohydrates, fat, fiber, and at least 6 distinctmetabolites including 2 distinct metabolites for modulating each one ofcholesterol, high-density lipoproteins, and low-density lipoproteins ina companion animal; wherein the metabolite that modulates cholesterol isselected from the group consisting of palmitoyl sphingomyelin, X-13215,1-stearoylglycerol (1-monostearin), alpha-tocopherol,7-alpha-hydroxycholesterol, 1-palmitoylglycerol (1-monopalmitin),7-beta-hydroxycholesterol, X-17010, glycerophosphorylcholine (GPC),X-16120, 2-linoleoylglycerophosphoethanolamine,5-methyl-2′-deoxycytidine, caproate (6:0),1-linoleoylglycerophosphoethanolamine, glycerol 3-phosphate (G3P),2-arachidonoylglycerophosphoethanolamine, glycerol 2-phosphate,dihydrocholesterol, pelargonate (9:0), stearamide,2-palmitoylglycerophosphoethanolamine, malate, heptanoate (7:0), andmixtures thereof; wherein the metabolite that modulates high-densitylipoproteins is selected from the group consisting of palmitoylsphingomyelin, X-13215, alpha-tocopherol, 1-stearoylglycerol(1-monostearin), 7-alpha-hydroxycholesterol, X-17010,1-arachidonoylglycerophosphocholine,2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol,1-palmitoylglycerol (1-monopalmitin),1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate(20:4n6), X-17612, 3-hydroxybutyrate (BHBA),2-arachidonoylglycerophosphocholine, pelargonate (9:0), stearamide,N2,N2-dimethylguanosine, 1-linoleoylglycerophosphocholine,dihydrocholesterol, X-11412, 1-stearoylglycerophosphocholine, X-17147,glycerate, heptanoate (7:0), 1-docosahexaenoylglycerophosphocholine,dihomo-linoleate (20:2n6), 2-linoleoylglycerophosphocholine,2-palmitoylglycerophosphocholine, and mixtures thereof; wherein themetabolite that modulates low-density lipoproteins is selected from thegroup consisting of 1-stearoylglycerol (1-monostearin), X-13215,palmitoyl sphingomyelin, alpha-tocopherol, 7-alpha-hydroxycholesterol,2-linoleoylglycerophosphoethanolamine, glycerophosphorylcholine (GPC),7-beta-hydroxycholesterol, 1-linoleoylglycerophosphoethanolamine,X-17010, 2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol(1-monopalmitin), caproate (6:0), 5-methyl-2′-deoxycytidine,2-arachidonoylglycerophosphoethanolamine, malate, glycerol 2-phosphate,glycerol 3-phosphate (G3P), 2-oleoylglycerophosphoethanolamine,N2,N2-dimethylguanosine, and mixtures thereof; and wherein the at least6 distinct metabolites are present in amounts that are at least one of(i) from about 0.01 weight % to about 10 weight % of the pet food foodcomposition or (ii) from about 1 IU to about 500,000 IU per kilogram ofthe pet food composition, wherein the pet food composition provides atleast a 5% increase or at least a 5% decrease in the amount of at leastone of cholesterol, high-density lipoproteins, or low-densitylipoproteins in the companion animal upon administration of the pet foodto the companion animal.
 2. The pet food composition of claim 1, whereinthe composition comprises at least 9 distinct metabolites, including atleast 3 distinct metabolites for modulating each one of cholesterol,high-density lipoproteins, and low-density lipoproteins in a companionanimal.
 3. The pet food composition of claim 1, wherein the compositioncomprises at least 12 distinct metabolites, including at least 4distinct metabolites for modulating each one of cholesterol,high-density lipoproteins, and low-density lipoproteins in a companionanimal.
 4. The pet food composition of claim 1, wherein the metabolitethat modulates cholesterol is selected from the group consisting ofpalmitoyl sphingomyelin, X-13215, 1-stearoylglycerol (1-monostearin),alpha-tocopherol, 7-alpha-hydroxycholesterol, 1-palmitoylglycerol(1-monopalmitin), 7-beta-hydroxycholesterol, X-17010,glycerophosphorylcholine (GPC), X-16120,2-linoleoylglycerophosphoethanolamine, 5-methyl-2′-deoxycytidine,caproate (6:0), and mixtures thereof.
 5. The pet food composition ofclaim 1, wherein the metabolite that modulates high-density lipoproteinis selected from the group consisting of palmitoyl sphingomyelin,X-13215, alpha-tocopherol, 1-stearoylglycerol (1-monostearin),7-alpha-hydroxycholesterol, X-17010,1-arachidonoylglycerophosphocholine,2-docosapentaenoylglycerophosphocholine, 7-beta-hydroxycholesterol,1-palmitoylglycerol (1-monopalmitin),1-docosapentaenoylglycerophosphocholine, caproate (6:0), arachidonate(20:4n6), and mixtures thereof.
 6. The pet food composition of claim 1,wherein the metabolite that modulates low-density lipoprotein isselected from the group consisting of 1-stearoylglycerol(1-monostearin), X-13215, palmitoyl sphingomyelin, alpha-tocopherol,7-alpha-hydroxycholesterol, 2-linoleoylglycerophosphoethanolamine,glycerophosphorylcholine (GPC), 7-beta-hydroxycholesterol,1-linoleoylglycerophosphoethanolamine, X-17010,2-palmitoylglycerophosphoethanolamine, 1-palmitoylglycerol(1-monopalmitin), and mixtures thereof.
 7. The pet food composition ofclaim 1, wherein the pet food composition provides at least a 10%increase in the amount of high-density lipoprotein in the companionanimal.
 8. The pet food composition of claim 1, wherein the pet foodcomposition provides at least a 10% decrease in the amount of at leastone of cholesterol and low-density lipoprotein in the companion animal.